TBX5 is a T-box transcriptional factor required for cardiogenesis and limb development. TBX5 mutations cause Holt-Oram syndrome characterized by congenital heart defects and upper limb deformations. Here we establish a novel function for TBX5 in pre-mRNA splicing, and we show that this function is relevant to the pathogenesis of Holt-Oram syndrome, providing a novel pathogenic mechanism for the disease. Proteomics in combination with affinity purification identifies splicing factor SC35 as a candidate TBX5-associating protein. Co-immunoprecipitation and glutathione S-transferase pulldown assays confirm the complex formation between TBX5 and SC35. TBX5 can bind to RNA homopolymers (polyribonucleotides) and to the 5'-splice site, which overrides the binding of SC35 to the same RNA. Overexpression of TBX5 increases the efficiency of pre-mRNA splicing and regulates alternative splice site selection. However, co-expression of TBX5 and SC35 antagonizes each other's positive effect on splicing. The most severe TBX5 mutation, G80R, with complete penetrance of the cardiac phenotype, strongly affects pre-mRNA splicing, whereas other mutations with incomplete penetrance of the cardiac phenotype, including R237Q, do not alter the splicing activity of TBX5. This study establishes TBX5 as the first cardiac gene and the first human disease gene with dual roles in both transcriptional activation and pre-mRNA splicing.